Dear sir,

I have problem when I use urea for solubulization. After dialysis the protein activity is lost.

It is not only your problem, indeed everyone in the world face this!

I would suggest, try different number of constructs with different fusion tags to improve the solubility and try with various expression host E.coli for ex:Rosetta,Rosetta Gami,pLys...etc.and then the growth and induction condition such as IPTG concentration,temperature(18deg-30deg) which plays a key role!

I got success by doing this! if nothing works then go for the process described above by renaturation.

Good luck,

Anand

Protocol is quite simple. But you need to optimize the temperature and IPTG concentration. Start with o.1mM IPTG. Take four flask 50 ml LB with appropriate antibiotics, keep two at 37 degree and two flask at 28 degree. Later on you can change from 28 to 18 degree variable temperature. Harvest your culture from 37 degree after 5 hours since you added 0.1 mM IPTG, of this one flask should be without IPTG for 5 hours. But for 28 degree keep it for 12 hours after IPTG induction, here too condition is same one would without IPTG. After you harvested, load the lysate and IB 37, Lysate and IB 28 degree, load the uninduced of the respective temperature accordingly. This will whether you are getting your protein in the lysate or not. Once your done let me know or also let me know if you havent understood the protocol.

You can try cell free protein expression system. It is a bit expensive, but I am sure you will get enough amount for your analysis. We did it before and it works fine with some (inclusion body making proteins in bacteria).

To avoid the formation of inclusion bodies or try to re-fold the inclusion bodies are difficult and complex task. There are a lot of protocols but it is necessary to have more information about why are you getting the inclusion bodies; are you expressing proteins from mammals, plant or bacteria? your recombinant proteins have His-tag? You can read this paper:

In vivo chaperone-assisted folding of alpha-1,6-fucosyltransferase from Rhizobium sp

Authors: Agatha Bastida, Montserrat Latorre, Eduardo García-Junceda

Chembiochem : a European journal of chemical biology. 07/2003; 4(6):531-3. (published)

Good luck

Some times decreasing the culture temperature before induction along with lower inducer concentration

also helps. for details check http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2374159/

Thanks every one for their kind suggestions

I have read that Co expression with heat shock e.coli also helps. Its great that everyone are sharing their experiences. it can be helpful to others also. I hope more suggestions continues......... thanks everybody

Try reducing the growing temperature to 30 degree. Induction with 0,1mM IPTG for 2-3 hours at 24 degree. And then try to purify. If it does not work, try 2% sarkosyl on your pellet (insoluble fraction) overnight. And then reduce it to 0,1-0,2% before loading into the beads, must also add Triton-X (4% [ratio with sarkosyl concentration]). Please see attachment.

http://www.ncbi.nlm.nih.gov/pubmed?term=Hu%20Tao%202010%20purifying

IPTG concentration and lower temperature combination is very successful criteria for insoluble recombinant proteins expression, which means a slow induction for longer period is essential. If this does not work well, then I would change the host E.coli cells for this purpose.

Carry out expression at low temperature.....u may find many articles on that

You might try to dissolve the inclusion bodies in 6M guinidinum Cl (GdmCl) followed by purification by reverse phase HPLC (e.g., C4, VYDAC column) and elution with an acetonitrile gradient. Lyophilize the fractions. Redissolve samples of each in 8M Urea and run on PAGE to access purity. Redissolve (lyophilized) fractions of interest in 6M GdmCl and dilute rapidly into a refolding buffer. Depending on your protein, you will get varying degrees of refolding.

Sometimes decreasing temperature or IPTG concentration helps to get soluble form of a protein. But, you should consider about the hydrophobicity of your protein, if it is a membrane protein, or surface protein then you have to truncate protein to get the soluble part, or use detergent during purification. If your protein is not a membrane protein then just dissolve inclusion in urea and refold it by dialysis.some proteins that are not recognised by E. coli during expression streight go to inclusion. if your protein is between 20-70 kDa and not much complex multimeric in structure, there are good possibilities to refold it by using urea.

Try also disruptingyour celles in à buffer with ARG GLU search google,this extract loose inclusin bodies and help to concentrate a prot for crystallography

Be sure that the expression condition of the protein is correct (temperature, pH, etc.). Good luck!

I´d second the lower temperature approach. Maybe adding the IPTG in low concentration with the feed instead of a bolus addition is helpful.

see A Simple Method for Improving Protein Solubility and

Long-Term Stability

Alexander P. Golovanov,*,† Guillaume M. Hautbergue,‡ Stuart A. Wilson,‡ and

Lu-Yun Lian*,†

Published on Web 07/01/2004